2.1. T Cell Growth Factors and Receptors
T cells secrete a variety of polypeptides affecting immunoregulation of hematopoietic cells and are themselves subject to regulation by hormone peptides interacting with specific receptors on their cell surface. Interleukin 2 (IL-2), originally termed T cell growth factor, is synthesized and secreted by antigen- or lectin-activated T lymphocytes in the presence of macrophage-derived interleukin-1 and must interact with specific high-affinity membrane receptors to exert its biological effects (Smith, K. A., 1980, Immunol. Rev., 51:337-357; Leonard, W. J., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:6957-6961). The interleukin 2 receptor (IL2R, Tac antigen) is not present on the surface of resting T or B lymphocytes. Upon activation by specific antigens or mitogens, T cell proliferation is mediated by an autocrine mechanism whereby activated cells secrete IL-2 and also express cell surface receptors for IL-2 (IL2R) (Leonard, W. J., et al., 1982, Nature, 300:267; Meuer, S. C., et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:1509). In addition to T cells, B cells (Mingari, M. C., et al., 1984, Nature 312:641-3; Pike, B. L., et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7917-21; Saiki, O., et al., 1988, J. Immunol. 140:853-8), NK cells (Ortaldo, J. R., et al., 1984, J. Immunol. 133:779-83; Kehrl, J. H., et al., 1988, J. Clin.. Invest, 81:200-5) and possibly monocytes (Herrmann, F., et al., 1985,,J. Immunol. 162:1111-6; Holter, W., et al., 1986,,J. Immunol. 136:2171-75) express a membrane-bound IL2R.
Current evidence suggests that both chains of the IL-2 heterodimer receptor expressed on the surface of activated T cells, are encoded by a single gene on human chromosome 10 (Leonard, W. J., et al., 1984, Nature 311:626:31). The high affinity IL2R that functions to signal T cell cycle progression is composed of two distinct polypeptide chains, each of which contains an IL-2 binding site (Teshigawara, K., et al., 1987, J. Exp. Med. 165:223). The larger IL-2 binding protein (75 kD molecular weight) is designated as the beta chain, whereas the smaller protein (55 kD molecular weight) is termed the alpha chain (Smith, K. A., 1988, Adv. Immunol. 42:165-78). The alpha chain was the first IL-2 binding protein to become recognized as an "activation antigen" on the surface of activated T cells (hence the name anti-Tac for "T activated") (Uchiyama, T., et al., 1981, J. Immunol. 126:1393-7).
Interaction of IL-2 with its cell surface receptor results in a continuous T cell proliferation (Greene, W. C. and Leonard, W. J., 1986, Ann. Rev. Immunol. 4:69-95; Smith, K. A., 1984, Ann. Rev. Immunol. 2:319-333). Measurement of IL2R provides information on the state of immune activation of the lymphoid population. This has been accomplished by measuring IL2R on cell surfaces using flow cytometry or fluorescence microscopy. Using monoclonal antibodies which define the IL-2 receptor, altered IL-2 receptor expression has been reported in a number of immune abnormalities (Greene and Leonard, supra; Depper, J. M., et al., 1984, J. Immunol. 133:1691-1695). Membrane IL2R has been found on certain B- or T-cell malignancies including Burkitt's lymphoma (Waldmann, T. A., et al., 1984, J. Exp. Med. 160:1450-1466), hairy cell leukemia (Waldmann et al., supra; Korsmeyer, S. J., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4522-4526), and human T cell leukemia virus (HTLV)-I-associated adult T cell leukemia (Depper, J. M., et al., 1984, J. Immunol. 133:1691-1695). The function of cellular IL2R in lymphoid malignancies has not been fully elucidated. Several cases of common, pre-B or T cell acute lymphoblastic leukemia (ALL) have been induced to express IL2R after in vitro activation (Touw, I., et al., 1985, Blood 66:556-561; Touw, I., et al., 1986, Blood 68:1088-1094; Matsuoka, M., et al., 1986, Leuk. Res. 10:597-603) and, in some cases, interleukin 2 stimulated subsequent colony formation of neoplastic progenitor cells in vitro (Touw, 1985, supra; Touw, 1986, supra).
Leukemia cells from some patients with T cell chronic lymphocytic leukemia were shown to have the receptors and a good proliferative response to exogenous interleukin 2 (Uchiyama, T., et al., 1985, J. Clin. Invest. 76:446-453; Tsudo, M., 1986, Blood 67:316-321). However, HTLV-1 associated adult T cell leukemia constitutively expressed high levels of cell surface IL2R but had no or very poor proliferative responses to interleukin 2 (Uchiyama, 1985, supra; Arya, S. K., et al., 1984, Science, 223:1086-1087). Ebert et al. (1985, Clin. Immunol. Immunopathol. 37:283-297) have reported that T cells from patients with AIDS virus lack the ability to express IL2R on their surface even when the cell is activated.
Utilizing immunohistochemical staining, Kurnick reported high numbers of IL2R and HLA-DR positive cells in lung tumor infiltrating lymphocytes (Kurnick, J. T., et al., 1986, Clin. Immunol. Immunopath. 38:367-380).
2.2. T Cell Surface Molecules
Clusters of differentiation (CD) have been established which define human leukocyte differentiation antigens (Bernard and Boumsell, 1984, Hum. Immunol. 11:1-10), by the comparison of reactivities of monoclonal antibodies directed against the differentiation antigens. The T cell surface antigens, their classification into epitope-defined subgroups, and their distributions on T cells have been studied by use of monoclonal antibodies directed against human T cells (Clark et al., 1983, Immunogenetics 18:599-615; Hansen et al., 1984, in Leucocyte Typing, Bernard, A., et al., eds., Springer-Verlag, New York, pp. 195-212). Some of the T cell clusters of differentiation and other T cell surface molecules are listed in Table I.
TABLE I __________________________________________________________________________ T CELL SURFACE MARKERS T Cell Molecular Detection Surface Weight Monoclonal Marker (kd) Expression Antibody Reference __________________________________________________________________________ T Cell 90 All T Cells T40/25 Brenner, M. B., Antigen et al., 1984, J. Receptor Exp. Med. 160: 541-551 CD8 30/43 Suppressor/ OKT8 Reinherz, E. L., et cytotoxic Leu 2 al., 1979, PNAS USA (subset of T 76:4061-4065; cells) Ledbetter, J. A., et al., 1981 Monoclonal Anti- bodies and T Cell Hybridoma Elsevier/ North Holland, N.Y., pp. 16-22. T6 49 Thymocytes & OKT6 Reinherz, 1979, Langerhans NAI/34 supra. Cells Leukemia Cells CD4 62 Helper/Inducer OK4 Kung, P. C., et al., Cells (subset Leu 3a 1979, Science 206: of T cells) 347-349 CD3 19 Pan T Cell OKT3 Kung, id. TAC 50 IL-2 Receptor Anti- Uchiyawa, T., et (Activated T TAC al., 1981, J. Cells) Immunol. 126(4):1393-1397 T9 94 Transferrin OKT9 Reinherz, E. L., Receptor et al., 1980, PNAS (Activated T USA 77:1588-1592 Cells) CD2 50 All T Cells OKT11 Verbi, W., et al., Leu 5 1982, Eur. J. Immunol. 12:81-86 VLA-1 130/165/210 Late Activated VLA-1 Helmer, M. E., et T Cells al., 1984, J. Immunol. 132:3011-3018 __________________________________________________________________________
These T cell surface markers serve as markers of the cell lineage, the identity of the functional T cell subset to which the T cell belongs, and the activation state of the T cell. Several of the cell surface molecules have been studied in great detail and have been found to be important in initiating and regulating immune functions, and are critical to communication processes between immune cells. T cell antigen receptor, a surface molecule which comprises a disulfide-linker dimer of approximately 90 kilodaltons (kd), recognizes specific antigens and is responsible for initiating a complex series of biochemical events which constitute the T cell activation process (Meuer, S. C., et al., 1984, Ann. Rev. Immunol. 2:23-50; Acuto, O., et al., 1983, Cell 34:717-726). The CD3 structure is a three-chain complex associated with the T cell receptor (Kannellopoulos, J. M., et al., 1983, EMBO J. 2:1807; Borst, J., et al., 1983, Eur. J. Immunol, 13:576; Van Den Elsen, P, et al., 1984, Nature 312:413; Meuer, S. C., et al., 1983, J. Exp. Med. 157:705). Lymphokine receptors, e.g. interleukin 2 (IL-2) receptor and interleukin 1 (IL-1) receptor, are essential for the activation and proliferation of T cells (Smith, K. A., 1984, Ann. Rev. Immunol. 2:319-333; Dower, S. K., et al., 1985, J. Exp. Med. 162:501-515). CD8 is a T cell specific surface glycoprotein expressed on the surface of approximately 30% of T lymphocytes associated with suppression and cytotoxic functions and the ability to recognize antigen in the context of class I MHC antigens (Swain, S. L., 1983, Immunol. Rev. 74:129-42) CD4 (OKT4 antigen) is a 55 kd glycoprotein expressed on the surface of approximately 60% of all T lymphocytes and is associated with helper function (Reinherz et al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:4061-4065) and the ability to recognize antigens in the context of type II MHC antigens (Swain, supra; Meuer, S. C., et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4395-99). CD4 has also been identified as the receptor for the HTLV-III virus associated with acquired immune deficiency syndrome (AIDS) (McDougal, J. S., et al., 1986, Science 231:382-385). These various cell surface markers have enormous clinical application potentials for the identification of lymphocyte populations and their functional status (Krensky, A. M. and Clayberger, C., 1985, Transplant. 39(4):339-348; Kung, P. C., et al., 1984, Monoclonal Antibodies in Clinical Investigations, Clinical Biochemistry-Contemporary Theories and Techniques, vol. 3, Academic press, pp. 89-115; Kung, P. C., et al., 1983, Int. J. Dermatol. 22(2):67-73).
Existing clinical methods of T cell typing involve the use of monoclonal antibodies which define T cell surface markers to detect the presence of specific cell surface markers on the T cell surface. Measuring the total numbers of T cells by surface markers has been useful for the characterization and classification of lymphoid malignancies (Greaves, M., et al., 1981, Int. J. Immunopharmac. 3(3):283-300). Changes in the relative percentage of T helper and T suppressor/cytotoxic cells were found to be associated with immune events in renal transplantation due to viral infection (Colvin, R. B, et al., 1981, Proc. 8th Int. Congr. Nephrol., Athens, pp. 990-996), autoimmune E. M., et al., 1981, Int. J. Immunopharmac. (3):313-319), and AIDS (Gupta, S., 1986, Clin. Immunol. Immunopathol. 38:93-100; Ebert, E. C., et al., 1985, Clin. Immunol. Immunopathol. 37:283-297).
The expression of T cell surface markers has also been used for the assessment of the immune status of patients. It has been established that by measuring the relative number of distinct, functional T cell subsets, and/or the relative number of activated T cells in peripheral blood or tissues, an assessment of the immunological condition of a patient is possible. The activation antigens (e.g. IL-2 receptor) appear to be involved in T cell growth and differentiation processes.
Antibodies to CD4 have been widely described (Kung, P. C., et al., 1979, Science 206:347-349) and are commercially available. A series of such antibodies reacting with non-competing epitopes on the CD4 molecule have been described. Such a set has been termed OKT4, OKT4A, OKT4B, OKT4C, OKT4D, OKT4E, and OKT4F (Rao, P. E., et al., 1983, Cell. Immunol. 80:310).
Antibodies directed against the CD4 or CD8 antigens have been shown to block cell function. Antibodies against CD4 block most helper T functions, mixed lymphocyte reactions and induction of T helper activity (Biddison, W. E., et al., 1984, J. Exp. Med. 159:783). Antibodies against CD8 block the cytotoxic activity of CD8 positive cytotoxic T lymphocytes (Swain, S L., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:7101-7105). Antibodies against CD4 have also been described that are capable of activating CD4-positive T cells. CD4 is internalized upon treatment of the cells with phorbol esters and resulting phosphorylation (Hoxie, J. A., et al., 1986, J. Immunol. 137:1194-1201).
The cloning of the gene encoding CD4 reveals that it, like CD8, is a member of the immunoglobulin supergene family, containing both amino acid (32%) and structural (.beta. sheets held together by disulfide bridges) homology at the V (variable)-like domain of CD4 to the V region of immunoglobulin (Maddon, P. J., et al., 1985, Cell 42:93-104). This V-like region of the molecule is followed by a stretch of 263 amino acids with no known homology to other molecules, followed by a transmembrane domain and highly charged cytoplasmic tail, containing serines which are phosphorylated upon activation (Littman, D. R., et al., 1984, Nature 325:453-55)
Homology between CD4 and CD8 is quite low. CD8 exists on the cell surface as dimeric or multimeric structures composed of a 33 kD monomer (Snow, P. M., et al., 1983, J. Biol. Chem. 258:14675-14681).